Patrick S Lown, Jessy J Cai, Seth C Ritter, Jacob J Otolski, Ryan Wong, Benjamin J Hackel
{"title":"在哺乳动物细胞的直接选择中,扩展的酵母表面显示连接体增强了配体的富集。","authors":"Patrick S Lown, Jessy J Cai, Seth C Ritter, Jacob J Otolski, Ryan Wong, Benjamin J Hackel","doi":"10.1093/protein/gzab004","DOIUrl":null,"url":null,"abstract":"<p><p>Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast-protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058008/pdf/gzab004.pdf","citationCount":"2","resultStr":"{\"title\":\"Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.\",\"authors\":\"Patrick S Lown, Jessy J Cai, Seth C Ritter, Jacob J Otolski, Ryan Wong, Benjamin J Hackel\",\"doi\":\"10.1093/protein/gzab004\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast-protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.</p>\",\"PeriodicalId\":54543,\"journal\":{\"name\":\"Protein Engineering Design & Selection\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2021-02-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058008/pdf/gzab004.pdf\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein Engineering Design & Selection\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1093/protein/gzab004\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein Engineering Design & Selection","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1093/protein/gzab004","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.
Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast-protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.
期刊介绍:
Protein Engineering, Design and Selection (PEDS) publishes high-quality research papers and review articles relevant to the engineering, design and selection of proteins for use in biotechnology and therapy, and for understanding the fundamental link between protein sequence, structure, dynamics, function, and evolution.